High-dose methylprednisolone mediates YAP/TAZ-TEAD in vocal fold fibroblasts with macrophages

The pro-fibrotic effects of glucocorticoids may lead to a suboptimal therapeutic response for vocal fold (VF) pathology. Targeting macrophage-fibroblast interactions is an interesting therapeutic strategy; macrophages alter their phenotype to mediate both inflammation and fibrosis. In the current study, we investigated concentration-dependent effects of methylprednisolone on the fibrotic response, with an emphasis on YAP/TAZ-TEAD signaling, and inflammatory gene expression in VF fibroblasts in physical contact with macrophages. We sought to provide foundational data to optimize therapeutic strategies for millions of patients with voice/laryngeal disease-related disability. Following induction of inflammatory (M(IFN/LPS)) and fibrotic (M(TGF)) phenotypes, THP-1-derived macrophages were seeded onto HVOX vocal fold fibroblasts. Cells were co-cultured +/−0.3–3000nM methylprednisolone +/− 3μM verteporfin, a YAP/TAZ inhibitor. Inflammatory (CXCL10, TNF, PTGS2) and fibrotic genes (ACTA2, CCN2, COL1A1) in fibroblasts were analyzed by real-time polymerase chain reaction after cell sorting. Ser211-phosphorylated glucocorticoid receptor (S211-pGR) was assessed by Western blotting. Nuclear localization of S211-pGR and YAP/TAZ was analyzed by immunocytochemistry. Methylprednisolone decreased TNF and PTGS2 in fibroblasts co-cultured with M(IFN/LPS) macrophages and increased ACTA2 and CCN2 in fibroblasts co-cultured with M(IFN/LPS) and M(TGF). Lower concentrations were required to decrease TNF and PTGS2 expression and to increase S211-pGR than to increase ACTA2 and CCN2 expression and nuclear localization of S211-pGR. Methylprednisolone also increased YAP/TAZ nuclear localization. Verteporfin attenuated upregulation of CCN2, but not PTGS2 downregulation. High concentration methylprednisolone induced nuclear localization of S211-pGR and upregulated fibrotic genes mediated by YAP/TAZ activation.


INTRODUCTION
The vocal fold (VF), an essential apparatus for phonation, vibrates hundreds of times a second. 1 Due to its role and anatomical location, the VF is inherently exposed to mechanical and environmental stress.
As many as 20 million people report voice disorders annually in the US. 2 In ammation is broadly associated with dysphonia and is likely etiologic for benign vocal fold lesion development.4][15] Optimizing GC therapy to minimize brosis while limiting in ammation has the potential to bene t millions of patients.
In the in ammatory milieu, dysfunctional cooperation between tissue-resident and in ltrated hematopoietic cells can drive pathological tissue responses, such as chronic in ammation and brosis. 168][19][20] Differential macrophage phenotypes are induced via exposure to stimuli through the shift from in ammatory to brotic environments. 17,21,22In ammatory stimuli, such as interferon-gamma (IFN-γ) and lipopolysaccharide (LPS), induce the in ammatory M1 phenotype. 17,234][25] However, various subtypes beyond the dualistic classi cation to the M1 and M2 are induced by individual stimuli, 17 likely related to organ-speci c responses to macrophages. 26,27For example, in VF broblasts, brotic genes were not activated by paracrine signaling from IL4-stimulated typical M2 macrophages, 27 which elicited a brotic response in non-VF broblasts. 28,29Conversely, TGF-β-stimulated macrophages induced a brotic response in VF broblasts.Independently, physical contact and paracrine signals from macrophages differentially activated VF broblasts. 27To that end, understanding interactions between VF broblasts and macrophages is foundational to optimally treat VF disease.
Recently, re nement of GC dose has emerged as a possible strategy to improve GC therapy. 15,30Our previous work with indirect co-culture models found brotic and in ammatory responses of VF broblasts triggered by macrophage-derived paracrine signals were promoted and inhibited by 'high' and 'low' concentrations of methylprednisolone, respectively. 31Based on this nding, we hypothesized minimizing GC concentrations to su ciently inhibit in ammation improves e cacy of GC therapy.However, previous co-culture studies employed a cell culture insert to allow only paracrine signaling.
Considering the in vivo environment in which macrophages directly engage broblasts, 32 co-culture models with direct intercellular communication further support the translation of in vitro ndings to support in vivo investigation.In addition, mechanisms underlying concentration-dependent negative and positive gene regulation remain unknown.
Based on the currently known biochemistry of GC signaling, unrelated to concentration-dependency, complex reactions of the GC receptor (GR) are thought to be a source of diverse GC functions. 10,33GR interacts with numerous proteins.The GC/GR complex binds and inhibits other transcription factors in the cytoplasm.Alternatively, GR translocated to the nucleus binds to both negative and positive gene regulatory elements.Various post-translational modi cations (phosphorylation, acetylation, SUMOylation) are involved in GR distribution and recruitment to gene regulatory elements.Additionally, accessibility to negative and positive gene regulatory elements is putatively altered by dimerization of GR concentrated in the nucleus. 105][36][37] In this pathway, Yes-associated protein (YAP) and transcriptional co-activator with PDZbinding motif (TAZ) are the core. 38Activated YAP/TAZ enters the nucleus and primarily serves as coactivators of TEA domain transcription factors (TEADs) to induce TEAD-dependent transcription.CCN2, a brotic gene induced by high-concentration GCs, is a target of YAP/TAZ-TEAD signaling; 39 this nding underlies the hypothesis that YAP/TAZ-TEAD signaling is speci cally activated by high-concentration GCs.
In the current study, a direct co-culture model was employed to further con rm concentration-dependent effects of methylprednisolone to alter brotic and in ammatory responses of human macrophages and VF broblasts.We additionally explored nuclear localization of GR and YAP/TAZ in this model to interrogate mechanisms underlying concentration-dependent effects of GCs.Ultimately, we seek to re ne GC therapy corroborated by mechanistic insight, to bene t millions of patients with voice-related disability.

RESULTS
Methylprednisolone altered in ammatory genes in direct co-culture of human VF broblasts and macrophages M(IFN/LPS) and M(TGF) stimulate in ammatory and brotic responses of VF broblasts. 27oncentration-dependent effects of methylprednisolone on gene expression were assessed using direct co-culture models of human VF broblasts with GFP-expressing M(IFN/LPS) and M(TGF) macrophages (G-M(IFN/LPS) and G-M(TGF)).In human VF broblasts co-cultured with G-M(IFN/LPS) macrophages, three in ammatory genes (TNF, PTGS2, and IL1B) were downregulated by methylprednisolone in a concentration-dependent manner.However, CXCL10, another in ammatory gene, was unchanged (Fig. 1).In co-culture with G-M(TGF), methylprednisolone decreased CXCL10 expression in VF broblasts and tended to inhibit expression of TNF, PTGS2, and IL1B.Methylprednisolone downregulated TNF, PTGS2, and IL1B in G-M(IFN/LPS) macrophages, and CXCL10 in G-M(TGF) macrophages in a concentration-dependent manner.In addition, TNF, PTGS2, and IL1B tended to decrease in response to methylprednisolone in G-M(TGF) macrophages.Collectively, IC 50 and IC 90 to decrease in ammatory genes were 1.8-3.3 and 6.8-27nM in VF broblasts (Table 1) and 2.4-11.6 and 7.7-36.8nM in macrophages (Table 2).

Methylprednisolone altered brotic gene expression in direct co-culture of human VF broblasts and macrophages
Methylprednisolone increased CCN2 and ACTA2 expression in VF broblasts directly co-cultured with G-M(IFN/LPS) and G-M(TGF) macrophages in a concentration-dependent manner (Fig. 2).However, COL1A1 expression was unchanged or decreased by methylprednisolone.CCN2 in G-M(IFN/LPS) and G-M(TGF) macrophages was concentration-dependently upregulated by methylprednisolone in the direct co-culture model.TGM2 and FN1, M2 markers associated with brosis, were also analyzed. 17,27,40The effect on FN1 expression was unclear.Weak tendencies of decreased and increased TGM2 were observed in response to methylprednisolone in G-M(IFN/LPS) and G-M(TGF) macrophages, respectively.

Ser211-phosphorylated GR was increased by low concentration methylprednisolone
Phosphorylation at Ser211 is thought to be most critical for ligand-induced activation of GR, as well as nuclear translocation. 41,42We performed Western blotting to assess altered Ser211-phosphorylated GR (S211-pGR), as well as total and Ser203-phosphorylated GR (tGR and S203-pGR).Directly co-cultured broblasts and macrophages were collected together and protein levels were assessed as mixtures of co-cultured cells.S211-pGR levels were increased by methylprednisolone regardless of macrophage phenotype and peaked around 10nM methylprednisolone (Fig. 3).This concentration was closer to the concentration required for gene downregulation than upregulation.However, S203-pGR, an inhibito of nuclear GR localization, 42 and tGR were decreased by methylprednisolone.Similar results were observed in separately collected broblasts and macrophages from indirect co-culture (Supplemental Figure S1).

Nuclear localization of Ser211-phosphorylated GR was increased by high concentration methylprednisolone
We subsequently performed immunocytochemistry to assess nuclear localization of GR.Regardless of macrophage phenotype, tGR-positive staining in DAPI-positive nuclear regions was concentrationdependently increased by methylprednisolone in both broblasts and macrophages in co-culture.tGR staining in nuclear regions peaked at 3-30nM methylprednisolone and decreased at higher concentrations (Fig. 4).S211-pGR in nuclear regions was also increased by methylprednisolone and plateaued at 100-300nM methylprednisolone.These ndings, collectively with qPCR data, suggested the concentration of methylprednisolone required to upregulate brotic genes was more related to S211-pGR nuclear localization than tGR.
Methylprednisolone induced Nuclear localization of YAP/TAZ in VF broblasts.Distribution of YAP/TAZ was assessed by immunocytochemistry.Regardless of the phenotype of cocultured macrophages, positive staining for YAP and TAZ increased in the nucleus of VF broblasts as methylprednisolone concentrations increased (Fig. 4).In contrast, methylprednisolone did not alter the distribution of YAP/TAZ in M(IFN/LPS) or M(TGF) macrophages.
Fibrotic gene expression induced by methylprednisolone was suppressed by inhibition of YAP/TAZ-TEAD signaling.
To assess involvement of YAP/TAZ in the negative and positive gene regulation of methylprednisolone, we pharmacologically inhibited YAP/TAZ-TEAD signaling in co-cultured broblasts in the presence of 30 or 1,000nM methylprednisolone.In VF broblasts co-cultured with M(IFN/LPS) macrophages, the decrease of TNF and PTGS2 by 30nM methylprednisolone was not reversed by vertepor n (Fig. 5).
However, increased CCN2, a target gene of YAP/TAZ-TEAD signaling, induced by 1,000nM methylprednisolone was inhibited by vertepor n.Vertepor n also inhibited ACTA2.Similarly, CXCL10 downregulation by 30nM methylprednisolone was not prevented by vertepor n in VF broblasts cocultured with M(TGF) macrophages, whereas increased CCN2 and ACTA2 induced by 1,000nM methylprednisolone was ameliorated by vertepor n.These ndings suggested CCN2 upregulation mediated by high-concentration GCs was driven with support of YAP/TAZ-TEAD signaling, but GCinduced suppression of those in ammatory genes was independent of YAP/TAZ-TEAD signaling.

DISCUSSION
GC therapy persists as a reasonable therapeutic option due to potent anti-in ammatory effects as well as the affordability and safety data accumulated over decades of clinical use.However, GCs have diverse functions and unfavorable side effects may negatively affect clinical outcomes.Optimizing GC therapy by reducing unfavorable effects could bene t millions of patients, while developing a novel therapeutic would also advance clinical care.The current study provided incremental data regarding concentration-dependent negative and positive gene regulation by methylprednisolone, as well as insight regarding mechanism(s) underlying this effect.
The IC 90 of methylprednisolone to reduce in ammatory gene transcription was lower than the EC 90 to promote brotic transcription in the current direct co-culture model; these data are similar to monocultured and indirectly co-cultured macrophages and VF broblasts. 30,31This nding further supports our hypothesis that reduced GC concentrations to a level su cient to inhibit in ammatory response is preferable to minimize brotic side effects. 15As noted in the previous indirect co-culture study, 31 the IC 90 range of methylprednisolone in the co-culture model was comparable to peak plasma levels of free methylprednisolone (21.1nM) after oral administration, 43,44 but the EC 90 values were lower than the concentration of methylprednisolone for injection (53-214mM). 45In vivo investigation is warranted to optimize GC dosing for e ciently inhibiting in ammation without activating brotic response.
The GR response to different concentrations of methylprednisolone was complex.Our data suggest concentration-dependent differential effects on GR phosphorylation/nuclear localization contribute to the complex biochemistry of GC-GR signaling.Although our data are insu cient to detail molecular events associated with different concentrations of GCs, some similarities were observed between methylprednisolone concentrations required to alter GR status and gene expression.Ideally, these data may be foundational to unravel the complexity related to GC concentration.Concentrations required to fully increase nuclear localization of S211-pGR were 100-300nM, comparable to the EC 90 for upregulation of brotic genes.In contrast, S211-pGR and nuclear localization of tGR reached a peak at 3-30nM methylprednisolone, close to the IC 90 for gene downregulation.Therefore, it seems the nuclear level of GR without Ser211-phosphorylation and/or cytoplasmic S211-pGR is related to negative gene regulation via low GC concentration, and the nuclear localization of S211-pGR is related to positive gene regulation via high GC concentration.Nuclear localization of GR and its recruitment to positive gene regulatory elements are restricted by various types of post-translational modi cations.These phenomena may explain why increased S211-pGR by low concentration methylprednisolone did not lead to increased nuclear accumulation or CCN2 upregulation.For example, phosphorylation at Ser203 and Ser226, and acetylation at Lys494 and Lys495 prevent nuclear localization and/or recruitment to positive gene regulatory elements. 10,42,46Regarding Ser203 phosphorylation, the function of Ser211 phosphorylation to drive nuclear localization of GR is likely overcome by Ser203 phosphorylation to prevent nuclear localization of GR. 42 On the other hand, recruitment of GR to negative gene regulatory elements, presumably associated with low-concentration methylprednisolone, reportedly requires SUMOylation at Lys293 of GR. 47 In addition, concentration of ligand-activated GR in the nucleus is thought to be crucial for GR dimerization and GR-mediated transcription via positive gene regulatory elements. 10,48,49In spite of our encouraging data, mechanisms underlying concentration-dependent negative and positive gene regulation are still unclear.
YAP/TAZ-TEAD signaling supports multiple brotic signaling pathways, such as SMAD, Wnt, and Rho. 50hibition of YAP/TAZ-TEAD signaling, as well as neutralization of connective tissue growth factor (encoded by CCN2), reduced the brotic response in multiple animal models and VF broblasts. 35,36,51,52n the current study, CCN2 upregulation induced by high-concentration methylprednisolone was reversed by vertepor n, but downregulation of CXCL10, TNF, and PTGS2 was not.This nding suggests the brotic response associated with YAP/TAZ-TEAD signaling is speci cally induced by high-concentration GCs.Blocking YAP/TAZ in combination with GC therapy may be another possible strategy to reduce brotic response induced by GCs.However, with regard to in ammation, YAP/TAZ reportedly has positive and negative roles dependent on cell types and organs. 50,53Notably, as shown in our immunocytochemistry results, YAP/TAZ and TEAD activities in hematopoietic cells are quite different from other cells. 50The impact of YAP/TAZ and TEADs in in ammatory responses of VF-resident and hematopoietic cells requires investigation to potentially target YAP/TAZ for VF diseases.
In conclusion, concentration-dependent differential effects of methylprednisolone were broadly observed across in vitro co-culture models of human VF broblasts and macrophages.S211-pGR nuclear localization and the activation of YAP/TAZ-TEAD signaling were likely associated with brotic gene expression mediated by high-concentration methylprednisolone in VF broblasts.

MATERIALS AND METHODS
The Supporting Information provides more detailed methodological speci cs.
Quantitative Real-Time Polymerase Chain Reaction (qPCR).HVOX broblasts and GFP-positive macrophages were separated by uorescence-activated cell sorting (FACS).RNA extraction, reverse transcription, and real-time polymerase chain reaction were performed using commercially available kits.Expression levels relative to GAPDH were quanti ed by the ΔΔCt method.
Western blotting and Immunocytochemistry.Following co-culture of broblasts and GFP-negative macrophages, Western blotting and immunocytochemistry were performed as described previously. 27,37tibodies are shown in Table S1   Western blots for S211-pGR, S203-pGR, and tGR.Human VF broblasts were directly co-cultured with M(IFN/LPS) or M(TGF) macrophages ± 0.1-3,000nM methylprednisolone for 24 hours.Proteins were extracted from the co-cultured cells without separating broblasts and macrophages.

Figures Figure 1
Figures

Table 1 and
55ata analysis.Data were collected from independently performed technical triplicate experiments, at least.The R drc package was employed on R studio to determine EC 50 , IC 50 , EC 90 , and IC 90 , and to t data into sigmoid curves, when applicable.55Simple dot plots are presented for other data.Means of EC x /IC x , as well as standard deviations, were calculated from the EC x /IC x estimations of triplicate experiments.Declarations RN, GJG, and RCB conceived the study.RN, MJG, and RB designed experiments.RN conducted most of the experiments and data analysis for gures 1-5 and tables 1 and 2. RB and GJG supported the experiments and data analysis for gures 1-5 and tables 1 and 2. RN wrote the manuscript.MJG and RCB supervised the study.2 are available in the Supplementary Files section.